title: Development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry for determination of amanitins in urine and liver samples.
abstract: Amanitins, highly toxic cyclopeptides isolated from various Amanita species, are the most potent poisons accounting for the hazardous effects on intestinal epithelium cells and hepatocytes, and probably the sole cause of fatal human poisoning. The present study was focused on the development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), following urine and liver sample preparation by protein precipitation with organic solvents, and solid phase extraction (SPE) procedure, for the determination of the amatoxins, α- and β-amanitin. Linearity, detection and quantification limits, selectivity, sensitivity, intra and inter-assay precision and recovery were studied, in order to guarantee reliability in the analytical results. The developed method proved to be specific and selective, with LOD (Limit of Detection) values for α- and β-amanitin of 0.22 and 0.20 ng mL(-1) in urine and 10.9 and 9.7 ng g(-1) in liver, respectively. LOQ (Limit of Quantification) values ranged from 0.46 to 0.57 ng mL(-1) in urine and 12.3-14.7 ng g(-1) in tissue, for both amanitins. Linearity, in the range of 10.0-200.0 ng mL(-1) or ng g(-1), shows that coefficients of correlation were greater than 0.997 for α-amanitin and 0.993 for β-amanitin. Precision was checked at three levels during three consecutive days with intra-day and inter-day coefficients of variation not greater than 15.2%. The extraction recovery presents good results for the concentrations analyzed, with values ranging from 90.2 to 112.9% for both matrices. Thus, the proposed analytical method is innovative, presents a high potential in the identification, detection and determination of α- and β-amanitins in urine and tissue samples, as well as in other biological samples, such as kidney and mushrooms.
authors.affiliation: CEF - Center for Pharmaceutical Studies, Health Sciences Campus, Pharmacy Faculty, University of Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal.
authors.forename: Marta
authors.initials: M
authors.lastname: Leite
authors.forename: Andreia
authors.initials: A
authors.lastname: Freitas
authors.forename: Anabela Marisa
authors.initials: AM
authors.lastname: Azul
authors.forename: Jorge
authors.initials: J
authors.lastname: Barbosa
authors.forename: Saul
authors.initials: S
authors.lastname: Costa
authors.forename: Fernando
authors.initials: F
authors.lastname: Ramos
mesh_terms: D000546:Amanitins / Q000032:analysis* / Q000097:blood / Q000652:urine*; D002623:Chemistry Techniques, Analytical / Q000379:methods* / Q000592:standards; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008099:Liver / Q000737:chemistry*; D015394:Molecular Structure; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization; D013997:Time Factors; D016482:Urinalysis / Q000379:methods*
chemical_list: D000546:Amanitins; C049197:beta-amanitin
keywords: Amanitin; Liver; Method validation; Tilmicosin; Urine